Methods of Detecting Analytes
Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3′ end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm 2 . Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3′ of the positional domain.
1 - 24 . (canceled)
25 . A method for determining the location of nucleic acid in a tissue section, the method comprising:
(a) providing an array comprising a plurality of features on a substrate, wherein a feature of the plurality of features comprises a plurality of capture probes immobilized thereon such that a capture probe of the plurality of capture probes has a free 3′ end, the feature occupying a distinct position on the array, and the capture probe comprising a nucleic acid molecule having the following domains oriented 5′ to 3′:
(i) a first sequence comprising a positional domain comprising a sequence unique to the feature; and
(ii) a second sequence comprising a capture domain comprising a nucleotide sequence complementary to at least a portion of the nucleic acid to be detected;
(b) placing the tissue section on the array such that the tissue section covers the feature on the array;
(c) hybridizing the nucleic acid from the tissue section to the capture domain of the capture probe, such that the nucleic acid is captured by the capture domain;
(d) generating a complementary DNA molecule by extending the free 3′ end of the capture probe using the captured nucleic acid as a ligation template, such that the generated complementary DNA molecule comprises a nucleotide sequence complementary to the positional domain;
(e) releasing the generated complementary DNA molecule, or a portion thereof, or generating and releasing a second strand complementary to the generated complementary DNA molecule, or a portion thereof, from the feature on the array, wherein the generated complementary DNA molecule, or the portion thereof, comprises the nucleotide sequence of the positional domain, or the second strand, or the portion thereof, comprises a sequence complementary to the nucleotide sequence of the positional domain; and
(f) identifying the nucleotide sequence of the positional domain present in the generated complementary DNA molecule or a complement thereof, wherein the nucleotide sequence of the positional domain, or the complement thereof, indicates that the generated complementary DNA molecule was obtained from nucleic acid in the tissue section at the distinct position where the tissue section covered the feature, thereby determining the location of the nucleic acid in the tissue section.
26 . The method of claim 25 , wherein step (e) comprises generating and releasing the second strand, or the portion thereof, from the feature on the array.
27 . The method of claim 26 , wherein the releasing of the second strand, or the portion thereof, from the feature on the array is performed by denaturation.
28 . The method of claim 26 , wherein the method further comprises a step of amplifying the second strand, or the portion thereof, released from the feature on the array.
29 . The method of claim 25 , wherein the method further comprises a step of amplifying the generated complementary DNA molecule to produce an amplified DNA molecule comprising the nucleotide sequence of the positional domain, or the complement thereof.
30 . The method of claim 29 , wherein the step of amplifying the generated complementary DNA molecule functions as the step of releasing the generated complementary DNA molecule, or the portion thereof, or the second strand, or the portion thereof, from the feature on the array.
31 . The method of claim 1 , wherein each capture probe consists of a nucleic acid molecule comprising the following domains oriented 5′ to 3′:
(i) a cleavage domain;
(ii) the first sequence comprising the positional domain; and
(iii) the second sequence comprising the capture domain,
wherein the step of releasing the generated complementary DNA molecule, or the portion thereof, or the second strand, or the portion thereof, from the feature on the array comprises cleaving the cleavage domain.
32 . The method of claim 31 , wherein the cleavage domain comprises a sequence that is cleaved by a cleavage enzyme.
33 . The method of claim 32 , wherein the step of cleaving the cleavage domain comprises using the cleavage enzyme that recognizes a nucleotide sequence in the cleavage domain and cleaves the generated complementary DNA molecule at a position that is 5′ to the positional domain, thereby releasing the generated complementary DNA molecule, or the portion thereof, or the second strand, or the portion thereof, from the feature on the array.
34 . The method of claim 25 , wherein the nucleic acid is RNA.
35 . The method of claim 34 , wherein the RNA is selected from the list consisting of:
mRNA, tRNA, rRNA, viral RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), ribozymal RNA, antisense RNA, and non-coding RNA.
36 . The method of claim 35 , wherein the RNA is mRNA and the capture domain is designed for the selective capture of mRNA.
37 . The method of claim 36 , wherein the capture domain that is designed for the selective capture of mRNA hybridizes to the poly-A tail of mRNA.
38 . The method of claim 37 , wherein the domain that is designed for the selective capture of mRNA comprises a poly-T sequence.
39 . The method of claim 25 , wherein the nucleic acid is genomic DNA.
40 . The method of claim 25 , wherein the capture domain comprises a random hexamer sequence or a random octamer sequence.
41 . The method of claim 1 , wherein step (f) comprises sequencing the generated complementary DNA molecule, or the portion thereof, or the second strand or the portion thereof, released from the feature on the array.
42 . The method of claim 41 , wherein the method further comprises:
imaging the tissue section after step (b); and
correlating sequence information obtained in step (f) with an image of the tissue section.
43 . The method of claim 42 , wherein the imaging is one or more of: light field, bright field, dark field, phase contrast, fluorescence microscopy, reflection, interference, and confocal microscopy.
44 . The method of claim 25 , wherein the plurality of capture probes are immobilized on the substrate by a chemical linker.
45 . The method of claim 25 , wherein the array is a bead array and the plurality of capture probes are immobilized on beads of the bead array.
46 . The method of claim 25 , wherein the method further comprises staining the tissue section.
47 . The method of claim 46 , wherein the staining comprises hematoxylin and eosin staining.
48 . The method of claim 25 , wherein the array comprises at least 1,000 features.
49 . The method of claim 25 , wherein the array comprises at least 50,000 features.
50 . The method of claim 25 , wherein the array comprises at least 100,000 features.
51 . The method of claim 25 , wherein the tissue section is a fixed tissue section.
52 . The method of 51 , wherein the fixed tissue section is a formalin-fixed paraffin-embedded tissue section.
53 . The method of claim 25 , wherein the capture probe comprises deoxynucleotides, ribonucleotides, peptide nucleic acids, or combinations thereof.
54 . The method of claim 25 , wherein the capture probe further comprises an amplification domain positioned 5′ to the positional domain.